The Microarray and Molecular Biology Module, housed in 741 square feet of space on the 5th and 2nd floors of the Kellogg Eye Center, facilitates vision research by providing a wide array of services and resources in the areas of microarray technology and molecular biology to the University's vision researchers and their collaborators within and outside the institution. This Module originated from the recent merger of the Molecular Biology Module and Genetics and DNA Microarray Module, resulting in improved efficiency and the addition of resources, techniques and services that were not previously offered by either Module. The Department of Ophthalmology and Visual Sciences plans to provide an additional 443 square feet of dedicated space for this Module on the 7th floor of the new building in early 2010 (see Appendix C). The MMB Module provides a comprehensive range of services required for microarray research, from experimental design, RNA isolation, and probe generation, to data analysis and confirmation of results by quantitative PCR. Staffing this Module is Matthew Brooks, a full-time research associate with extensive experience in both molecular biology and microarray methods. Mr. Brooks assists investigators with RNA isolation techniques, including from samples excised by microdissection via the Module's Arcturus AutoPix Laser Capture Microdissection system. The use of Affymetrix GeneChips by our vision scientists for gene profiling research is facilitated by the availability of an Affymetrix GeneChips Fluidic Station 450, an Affymetrix GeneChip Hybridization Oven 640, an Affymetrix GeneChip Scanner 3000, and an Affymetrix 428 Array Scanner. Mr. Brooks assists investigators with probe generation, GeneChip hybridization, data acquisition, and data analysis. In addition to the standard Affymetrix protocol, more recent techniques allowing probe generation using total RNA amounts as low as 5 ng are also available. These non-traditional techniques of probe generation have proved invaluable to vision researchers here at the University of Michigan and their collaborators in generating gene expression data from limited starting material, such as isolated photoreceptors and Muller cells. In addition to Affymetrix arrays, the MMB Module has considerable expertise in generation of custom microarrays of cDNA clones or amplified promoter regions. Such custom arrays offer unique avenues of investigation, i.e., a focus on a specific set of functional genes or those not present on Affymetrix GeneChips. The Module has produced custom cDNA arrays of human, mouse and zebrafish retinal genes as well as human RPE genes for various investigators and for different projects. Efforts are underway to generate arrays of amplified DNA fragments corresponding to the promoter regions of "eye" genes, which have significant promise for ChIP (chromatin immunoprecipitation)-on-Chip studies that seek to identify combinatorial gene regulatory networks. With such capabilities, options are endless for generating custom arrays that are developed to investigator specifications. All the basic microarray analysis methods, including normalization of data, generation of lists of present/absent genes, and lists of differentially expressed genes, are performed in-house by Ritu Khanna, M.S., a computer scientist and software engineer by training who is available on a part-time basis. More advanced statistical analysis is available through collaboration with Dr. Swaroop and Dr. Alfred Hero from the Electrical Engineering and Computer Science Department. This team has developed statistical methods for analysis and clustering of microarray data, including FDR-CI, Pareto, and network constrained clustering, which are being used extensively. New methods are being pursued to construct gene regulatory networks and pathways. Another major function of the MMB Module is to provide access to and training for small and large equipment that would be impractical for a single laboratory to acquire or maintain. Mr. Brooks is responsible for maintaining all of the Module's equipment and training research personnel to ensure proper use. The available equipment include: a Molecular Dynamics Storm 840 Phosphoimager, an Affymetrix GeneChip Scanner 3000, an Affymetrix 428 Array Scanner, an Affymetrix GeneChips Fluidic Station 450, an Affymetrix GeneChip Hybridization Oven 640, a Kodak X-OMAT 1000A film developer, two Kodak 440CF Image Stations, two US Filter Elga Purelab Plus water purification systems, a Beckman J2-21M centrifuge, a Beckman L8-70M ultra centrifuge, a Beckman Optima L-90K ultra centrifuge, 2 Integrated SpeedVac ISS110 systems, a Tuttnauer- Brinckman 3870E tabletop autoclave, an Arcturus AutoPix Laser Capture Microdissection system, a Molecular Devices SpectraMax 190 microplate reader, a Beckman DU530 spectrophotometer, a Perkin-Elmer Lambda Bio20 spectrophotometer, a Thermo Spectronic French Press, a Beckman 5500 gamma counter, a Perkin- Elmer WinSpectral 1414 scintillation counter, an Alpha Innotech Image Station, a Turner BioSystems Veritas microplate luminometer, an Amaxa Nucleofactor II Transfection System, and a Virtek SDDC-2 robotic microarrayer. Finally, the MMB Module facilitates the research of vision scientists by providing a variety of services including gene cloning consultation, primer design, DMA modification protocols, sequence analysis, and database services. These protocols have assisted participating investigators in the construction of cDNA libraries, gene expression cassettes, and bait/prey vectors for yeast two-hybrid assays. Recombinant protein expression using the Baculovirus Expression System (BBS) is also available, allowing the production of large quantities of post-translationally modified recombinant protein. The expertise to produce and purify His-Tag and GST fusion proteins also exists in-house. These services can advance functional studies, including the development of protein activity assays, crystal structure analysis, protein modification analysis, and antibody development.